Silica particles are, by far, the most widely used supports for reversed-phase liquid chromatography stationary phases. The high mechanical stability, monodisperse particles, high surface area, and easily tailored pore size distributions make silica superior to other supports in terms of efficiency, rigidity, and performance. Silica bonding chemistry also allows for a wide variety of stationary phases with different selectivities to be made on silica.
Silanes are the most commonly used surface modifying reagents in liquid chromatography. For example, An Introduction to Modern Liquid Chromatograhy, Chapter 7, John Wiley & Sons, New York, N.Y. 1979; J. Chromatogr. 352, 199 (1986); J. Chromatogr. 267, 39 (1983); and Advances in Colloid and Interface Science 6, 95 (1976) each disclose various silicon-containing surface modifying reagents.
Typical silane coupling agents used for silica derivatization have the general formula EtOSiR1R2R3 or ClSiR1R2R3, where each R represents organic groups, which can differ from each other or all be the same. For reversed-phase chromatography, the silane coupling agent has traditionally been
—Si(CH3)2(C18H37), where C18H37, an octadecyl group, yields a hydrophobic surface. The reaction, when carried out on the hydroxylated silica, which typically has a maximum surface silanol concentration of approximately 8 μmol/m2, does not go to completion due to the steric congestion imposed by the octadecyl groups on the coupling agent.
To improve the quality of the original chemically bonded phase by blocking access to some residual silanol groups on the silica surface, the bonded phase is usually further endcapped using small organic silanes. The endcapping is usually carried out with compounds able to generate trimethylsilyl groups, (CH3)3—Si—, the most popular being trimethylchlorosilane (TMCS) and hexamethyldisilazane (HMDS). The majority of free surface silanols, which are under the dimethyloctadecylsilyl groups, cannot react with the endcapping reagents because of steric hindrance. In the traditional endcapping step, only ˜0.2 μmol/m2 surface silanol groups are bonded based on the carbon loading data. The highest coverage attained in laboratory studies has been ˜4.5 μmol/m2, while the coverage available in commercial chromatography columns is much less, usually on the order of 2.7-3.5 μmol/m2, even after endcapping.
These residual surface silanols interact with basic and acidic analytes via ion exchange, hydrogen bonding and dipole/dipole mechanisms. However, this secondary interaction between analytes and residual silanol groups results in increased retention times, excessive peak tailing, especially at mid pH range for basic compounds, and irreversible adsorption of some analytes.
To overcome the problems of residual silanol activity, many methods have been tried such as the use of ultrapure silica, the use of carbonized silica, the coating of the silica surface with polymeric compositions, the endcapping of residual silanol groups, and the addition of suppressors such as long chain amines to the eluent. In practice, however, none of these approaches has been totally satisfactory. A general review of silica support deactivation is given by Stella et al. [Chromatographia (2001), 53, S-113-S115].
A method to eliminate surface silanols by extreme endcapping is described in U.S. Pat. No. 5,134,110. While traditional endcapping can physically bond some residual silanol groups, at least 50% of the surface silanols remain unreacted. U.S. Pat. No. 5,134,110 describes an endcapping method for octadecyl-silylated silica gel by high temperature silylation. Polymeric chemically bonded phases originated from trichlorosilanes were endcapped using hexamethyldisilazane or hexamethylcyclo-trisiloxane at very high temperature, above 250° C., in a sealed ampoule. The resulting endcapped phases were shown to perform with excellence on the Engelhardt test. This result was explained by the formation of dimethylsilyl loop structures on the surface, leading to the elimination of silanols. This method had the disadvantage in that it was used on a polymeric phase, and polymeric phases usually have poor mass transfer and poor reproducibility. Also the high temperature of silylation in a sealed ampoule is not practical and difficult to perform commercially, as compared with the traditional liquid phase endcapping procedure.
Another method of reducing the effect of surface silanols is to introduce polar embedded groups in the octadecyl chain. These embedded groups, generally contain nitrogen atoms and amides such as disclosed in European Patent No. EP0397301 and carbamates such as disclosed in U.S. Pat. No. 5,374,755. Most recently, urea groups have been shown to reduce the undesirable silanol interactions. Phases with incorporated polar groups clearly exhibit lower tailing factors for basic compounds, when compared with traditional C-18 phases. Some mechanisms have been proposed, while some evidence leads to the belief that the surface layer of an embedded polar group phase should have a higher concentration of water due to the hydrogen bonding ability of the polar groups near the silica surface. This virtual water layer suppresses the interaction of basic analytes with residual surface silanols and permits separation with mobile phase having 100% water.
A disadvantage of this approach is that the presence of the water layer seems to contribute to a higher dissolution rate of the silica support, as compared to their alkyl C-8 and C-18 counterparts. In a systematic column stability evaluation, an embedded amide polar stationary phase was shown to be less stable. This result may be predictable, due to the higher water content near the underlying silica surface for polar embedded phases. The embedded polar groups also cause adsorption of some analytes when the phases are hydrolyzed or the phases are not fully reacted during phase preparation, leaving amine or hydroxyl groups on the surface. For example, the hydrolyzed amide phase leaves aminopropyl moieties on the surface, that strongly adsorb acidic and polar compounds, causing their peaks to be tailed, or to be missing completely.
The polar embedded phases are also more hydrophilic than the traditional C-18 phases, enhancing the retention of polar compounds, whereas the retention of hydrophobic analytes is much less on polar-embedded columns than on the traditional C-18 columns. As a result, the phase selectivity is quite different from traditional C-18, which causes a change in the order in which analytes elute relative to each other from the column. Consequently, methods developed on traditional C-18 columns cannot be transferred to polar embedded phase columns.
Another method for reducing the effect of surface silanols is to use a phase that can sterically protect surface silanols. U.S. Pat. No. 4,705,725 to Du Pont discloses bulky diisobutyl (with C-18) or diisopropyl (with C-8, C3, phenyl propyl, cyano propyl) side chain groups (Zorbax® Stable Bond reversed-phase columns) that stabilize both long and short chain monofunctional ligands and protect them from hydrolysis and loss at low pH. The bulky side groups increase the hydrolytic stability of the phase. Such a moiety is less vulnerable to destruction at low pH, and better shields the underlying silanols. The sterically protected phases are extremely stable at low pH. The sterically protected silane phases are not endcapped; therefore, the loss of small, easily hydrolyzed endcapping reagents under acidic mobile phase condition is avoided. At pH<3, the phase has excellent performance, in terms of peak shape, retention, reproducibility, and lifetime. In this pH range, the silanol groups on a type B silica are nearly completely protonated, and as a result, they do not act as sites for secondary interaction. The coverage density is, however, much lower than for dimethyl ODS phases. The ligand density of diisobutyloctadecyl phase is ˜2 μmol/m2 when compared to the conventional dimethyloctadecyl phase with a ligand density of 3.37 mmol/m2.
U.S. Pat. No. 5,948,531 discloses the use of bridged propylene bidentate silanes or a bidentate C-18 phase (Zorbax® Extend-C-18 columns), to restrict analyte access to residual silanols by incorporating a propylene bridge between two C-18 ligands. The bidentate C-18 phase retains the benefits of monofunctional silane phases (high column efficiency, reaction repeatability) while demonstrating good stability in high and low pH mobile phases. Zorbax Stable-Bond C-18 (SB-C-18) and Zorbax Extend-C-18 columns also have very similar selectivity to the traditional C-18 columns.
Basic compounds appear in widely divergent areas, such as the environmental, chemical, food, and pharmaceutical industries. In the latter, in particular, over 80% of commercialized drugs are estimated to possess a basic function. Therefore, it is of crucial importance to develop practical HPLC stationary phases having minimized surface silanol activity.